scholarly journals Adenovirus vector–mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane

2002 ◽  
Vol 9 (11) ◽  
pp. 897-907 ◽  
Author(s):  
Rachel L Cowen ◽  
Judith C Williams ◽  
Steve Emery ◽  
David Blakey ◽  
John L Darling ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Adenovirus Vector ◽  
Converting Enzyme ◽  
High Level Expression ◽  
Carboxypeptidase G2 ◽  
High Level

scholarly journals High level expression of transfected G protein αi3subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts

FEBS Letters ◽  
1992 ◽  
Vol 312 (2-3) ◽  
pp. 223-228 ◽  
Author(s):  
Sylvie Hermouet ◽  
Philippe de Mazancourt ◽  
Allen M. Spiegel ◽  
Marilyn Gist Farquhar ◽  
Bridget S. Wilson
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Membrane Targeting ◽  
High Level Expression ◽  
3T3 Fibroblasts ◽  
High Level ◽  
Nih 3T3

scholarly journals Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

1992 ◽  
Vol 12 (1) ◽  
pp. 422-432 ◽  
Author(s):  
M Tal ◽  
B Thorens ◽  
M Surana ◽  
N Fleischer ◽  
H F Lodish ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Glucose Transporter ◽  
Islet Cells ◽  
T Antigen ◽  
High Level Expression ◽  
Glut 1 ◽  
Secondary Tumors ◽  
Low Levels ◽  
High Level

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.

scholarly journals Inducible Overexpression of a Toxic Protein by an Adenovirus Vector with a Tetracycline-Regulatable Expression Cassette

1998 ◽  
Vol 72 (3) ◽  
pp. 2289-2296 ◽  
Author(s):  
Bernard Massie ◽  
France Couture ◽  
Linda Lamoureux ◽  
Dick D. Mosser ◽  
Claire Guilbault ◽  
...  
Keyword(s):  
Cell Lines ◽  
Recombinant Proteins ◽  
Recombinant Adenovirus ◽  
Adenovirus Vector ◽  
Expression Cassette ◽  
Cell Protein ◽  
Animal Cells ◽  
High Level Expression ◽  
Simplex Virus ◽  
High Level

ABSTRACT We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(Δ2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce ΔR1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5ΔR1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy.

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice

1992 ◽  
Vol 12 (1) ◽  
pp. 422-432
Author(s):  
M Tal ◽  
B Thorens ◽  
M Surana ◽  
N Fleischer ◽  
H F Lodish ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Glucose Transporter ◽  
Islet Cells ◽  
T Antigen ◽  
High Level Expression ◽  
Glut 1 ◽  
Secondary Tumors ◽  
Low Levels ◽  
High Level

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.

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